KR20170084155A - Activated carbon for the removal of leachables and/or extractables - Google Patents

Abstract

The present invention relates to the purification of target molecules such as recombinant and / or biotherapeutic proteins. Activated carbon can be used to remove leachates and / or extracts resulting from disposable devices used in the process.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to an activated carbon for removing leachate and /

The present invention relates to the purification of target molecules such as, for example, recombinant and / or biotherapeutic proteins produced from a cell expression system. Activated carbon can be used to remove leachates and / or extracts resulting from disposable devices used in the process.

The efficient and economical large-scale preparation of therapeutic proteins, including biomolecules, such as antibodies, peptides or hormones, is an increasingly important consideration in the biotechnology and pharmaceutical industries. Generally, purification methods are very sophisticated, expensive, and involve many different steps.

Typically, proteins are produced using cell culture methods, for example, using recombinantly engineered mammalian or bacterial cell lines to produce the desired protein. In general, the isolation of target proteins from one or more impurities, such as, for example, host cell proteins, media components, and nucleic acids after expression of the target protein, presents a tremendous challenge. This separation and purification is particularly important when the therapeutic protein is intended for human use and must be approved by regulatory agencies such as the United States Food and Drug Administration (FDA).

Conventional methods for purification of proteins today often include at least the following steps: (a) purification steps for cell and cell debris removal using, for example, fractional centrifugation and / or filtration; And (b) one or more downstream chromatographic steps for separating the desired protein from the various impurities in the purified cell culture feed.

As a result, the production and purification of biomolecules is a multi-step procedure involving several types of purification media and also various types of apparatus.

Emerging trends in the biopharmaceutical industry exist that use single use and / or disposable plastic materials for manufacturing flexible, safe, reduced capital and operational costs. Disposable bioprocessing instruments are available in a wide range and scale from solution containers, transfer tubing to various devices. These instrument components are in direct contact with the product during the manufacturing process. It has been found that leachates and / or extracts from plastic materials can enter the final product (W. Ding, Chemie Ingenieur Technik 2013, 85, No. 1-2, 186-196). Although levels of leachate / extract are usually lower than those provided by industry guidelines (Q3C), low levels of leachate / extract also potentially affect drug product safety and lead to performance, resulting in product safety and quality problems. There is a concern that it can be done.

Consequently, there is a need to remove the leachate / extract from the target biomolecules in the bioprocess to ensure drug safety and quality during use of the disposable device.

Brief Description of the Invention

It has been found that the amount of leachate / extract in the target molecule formulation resulting from the use of a disposable device can be removed or reduced by filtration through an activated carbon device.

Accordingly, the present invention relates to a method of purifying target molecules involving the use of disposable devices whereby the target molecules are separated from the leachates and / or extracts using activated charcoal. Typically, tablets are made by contacting the activated carbon with a target molecule and potentially a liquid comprising leachate and / or extract, whereby the leachate and / or the extract are kept on contact with activated carbon or bound on activated carbon, The liquid containing the molecules is not bound and is then separated from the activated carbon by, for example, filtration.

In one embodiment, the target molecule is a protein having a molecular weight of 10,000 or more.

In one embodiment, the target molecule is a glycoprotein.

In one embodiment, the target molecule is an antibody.

In a preferred embodiment, the leachate and / or extract is one or more of the following components: acetaldehyde, toluene, 2-hexanone, acetone, 2-butanone, ethyl acetate, bisphenol A, benzyl alcohol, trimethylsilanol, formaldehyde , 2-ethylhexyl phthalate, 1-methylethyl ester acetic acid, 2,4-di-t-butyl phenol, 2-octanone, 2-pentanone, 3,3- Butanol, butanol, cyclohexanone, ethanol, formic acid, heptaethylene glycol, hexanal, methyl isobutyl ketone, pentanal, t-butanol, Tetrahydrofuran, methyl formate.

In a preferred embodiment, the method is carried out at a pH of from 3 to 9.

In another preferred embodiment, the activated carbon is an activated polymer obtained by pyrolysis of an organic polymeric material, preferably polystyrene.

In a preferred embodiment, the activated carbon has a median particle size of 5 to 40 [mu] m.

In one embodiment, the target molecule is separated from the leachate and / or extract by filtration through one or more filters comprising activated carbon.

In one embodiment, the method of the invention is used in a manufacturing and / or purification process comprising one or more of the following steps:

- Cell culture in a bioreactor

- purification

- refine

- percolation

In another embodiment, in the method of the invention, the target molecule is used in a disposable device wherein the disposable device is used in combination with a mechanical deformation of the disposable device at a temperature in excess of < RTI ID = 0.0 > 25 C ≪ RTI ID = 0.0 > activated carbon. ≪ / RTI >

The invention also relates to the use of activated carbon for the removal of leachates and / or extracts from liquids.

In a preferred embodiment, the liquid is contacted with activated carbon in a filtration device, whereby the liquid flows through a filtration device comprising activated carbon such that the leachate and / or extract is bound to activated carbon, Is not bonded to activated carbon.

Figure 1 shows the adsorption of the reference to the toxicity and the lower toxicity for the selected material.
Figure 2 shows the adsorption of a selected reference to various materials.
Figure 3 shows the adsorption of ethyl acetate on the selected activated carbon material.
Figure 4 shows the results of mass spectrometry using the Electrospray Ionization (ESI +) method for extract / leach detection after disposable assembly and after loading the treated water into a glass column packed with activated carbon.
Figure 5 shows the results of mass spectrometry using the Electrospray Ionization (ESI +) method for extract / leach detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Figure 6 shows the results of mass spectrometry using the Electrospray Ionization (ESI-) method for extract / leach detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Figure 7 shows the results of mass spectrometry using the atmospheric pressure chemical ionization (APCI-) method for extract / leach detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Figure 8 shows the results of mass spectrometry using the atmospheric pressure chemical ionization (APCI-) method for extraction / leachate detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Figure 9 shows the results of mass spectrometry using the Electrospray Ionization (ESI +) method for extract / leach detection after disposable assembly and loading the treated water into a glass column packed with activated carbon.
Figure 10 shows the results of mass spectrometry using the Electrospray Ionization (ESI +) method for extraction / leachate detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Figure 11 shows the results of mass spectrometry using the Electrospray Ionization (ESI +) method for extraction / leachate detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Figure 12 shows the results of mass spectrometry using the atmospheric pressure chemical ionization (APCI-) method for extract / leach detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Figure 13 shows the results of mass spectrometry using the atmospheric pressure chemical ionization (APCI-) method for extraction / leachate detection after disposable assembly and after loading the treated water into the Millistak + ® filter.
Further details regarding the drawings can be found in the examples.

Justice

Before describing the invention in detail, it is to be understood that the invention is not limited to the particular composition or method steps, but may vary by itself. It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "ligand" includes a plurality of ligands, and reference to "antibody" includes a plurality of antibodies and the like.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The following terms are defined for the purposes of the present invention as described herein.

The term "target molecule" as used herein refers to any molecule, material or compound that must be isolated, separated or purified from one or more other components of the sample, e.g., impurities. Examples of target molecules are the group of antibodies, fragment antigen binding (Fab), fragment constant (Fc), proteins, peptides, recombinant proteins, other natural compounds, other biopharmaceutical compounds, vaccines or biopharmaceutical compounds. In a preferred embodiment, the target molecule is a biomolecule, preferably a protein. In a highly preferred embodiment, the target molecule is an antibody. In manufacturing and / or purification methods, the target molecule is typically present in the liquid. The liquid may be water, buffer, non-aqueous solvent such as ethanol or any mixture thereof. In addition to the target molecule, the liquid may contain one or more impurities. The composition of the liquid may vary during manufacture and / or purification depending on the method steps being performed. After the chromatographic step, due to the eluent used in the chromatography step, the liquid typically contains a different solvent than before. Typically, only after the last purification step, the target molecule can be dried for the production of the final dosage form.

The term "antibody" refers to a protein having the ability to specifically bind to an antigen. Typically, the antibody has a basic four-polypeptide chain structure consisting of two heavy chains and two light chains, which chain is stabilized by, for example, interchain disulfide bonds. The antibody may be a monoclonal or polyclonal, and may be in monomeric or polymeric form, for example the IgM antibody is in the form of a dimer and / or the IgA antibody is in monomeric, dimeric or multimeric form . The antibody may also comprise antibody fragments and multispecific antibodies (e. G., Bispecific antibodies) if they are maintained or modified to include ligand-specific binding domains. The term "fragment" refers to a portion or portion of an antibody or antibody chain that contains less than amino acid residues than a complete or complete antibody or antibody chain. Fragments may be obtained through chemical or enzymatic treatment of a titer or complete antibody or antibody chain. Fragments can also be obtained by recombinant methods. When produced by recombination, the fragment can be expressed alone or as part of a larger protein called a fusion protein. Exemplary fragments include Fab, Fab ', F (ab') 2, Fc and / or Fv fragments. Exemplary fusion proteins include Fc fusion proteins. According to the present invention, the fusion protein also includes the term "antibody ".

In some embodiments, the antibody is an Fc region containing protein, such as an immunoglobulin. In some embodiments, the Fc region containing protein is a recombinant protein comprising an Fc region of an immunoglobulin fused to another polypeptide or fragment thereof. Exemplary polypeptides include, for example, renin; Growth hormone (including human growth hormone and bovine growth hormone); Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipid protein; alpha-1-antitrypsin; Insulin a-chain; Insulin beta -chain; Proinsulin; Follicle stimulating hormone; Calcitonin; Luteinizing hormone; Glucagon; Coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; Anticoagulants such as protein C; Atrial sodium diuretics; Pulmonary surfactants; Plasminogen activators such as urokinase or human urine or tissue-type plasminogen activator (t-PA); Bombesh Shin; Thrombin; Hematopoietic growth factors; Tumor necrosis factor-a and -beta; Enkephalinae; RANTES (regulatory normal T-cell expression and secretion upon activation); Human macrophage inflammatory protein (MIP-1-a); Serum albumin, such as human serum albumin; Muellerian - inhibitory substance; Alelaxacin a-chain; Lelactin beta -chain; Prolelaxacin; Mouse gonadotropin-related peptides; Microbial proteins such as? -Lactamase; DNase; IgE; Cytotoxic T-lymphocyte-associated antigen (CTLA) (e.g., CTLA-4); Inhivin; Actibin; Vascular endothelial growth factor (VEGF); Receptors for hormones or growth factors; Protein A or D; Rheumatic factor; Neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5 or NT-6) Nerve growth factors such as NGF-β .; Platelet-derived growth factor (PDGF); Fibroblast growth factors such as? FGF and? FGF; Epithelial growth factor (EGF); Transforming Growth Factor (TGF) such as TGF-alpha and TGF-beta (including TGF-beta 1, TGF- beta 2, TGF- beta 3, TGF- beta 4, or TGF- beta 5); Insulin-like growth factors-I and -II (IGF-I and IGF-II); Death (I-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein (IGFBP); CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; Erythropoietin; Bone conduction factor; Antibody toxin; Bone morphogenetic protein (BMP); Interferons such as interferon-a, -beta, and-gamma; Colony stimulating factors (CSF) such as M-CSF, GM-CSF, and G-CSF; Interleukin (IL), such as IL-I to IL-IO; Superoxide disutase; T-cell receptors; Surface membrane protein; Corrosion promoting factor; Viral antigen, e. G., Part of the AIDS envelope; Transport protein; Otic receptor; Addressin; Regulatory proteins; Integrins such as CDl Ia, CDl Ib, CDl Ic, CD 18, ICAM, VLA-4 and VCAM; Tumor-associated antigens such as HER2, HER3 or HER4 receptors; And fragments and / or variants of any of the listed polypeptides. In addition, an antibody according to the present invention is any protein or polypeptide, fragment or variant thereof, which is specifically bound to any one of the listed polypeptides.

As used herein and unless otherwise stated, the term "sample" refers to any composition or mixture comprising a target molecule. The sample may be of biological or other source origin. Biological sources include eukaryotic and prokaryotic sources such as plant and animal cells, tissues, and organs. Preferred samples include cell culture fluids such as mammalian cell cultures such as CHO, NS-0, SP2 / 0, BioWa, bacterial cell cultures such as E. coli, B. subtilis, Water, or filamentous fungus. The sample may also contain diluents, buffers, detergents, and contaminants, debris, and the like that are found mixed with the target molecule. The sample may be "partially purified" (e. G., Applied to one or more purification steps, such as filtration steps), or may be obtained directly from a host cell or organism producing the target molecule Cell culture fluid).

As used herein, the term "impurity" or "contaminant" refers to a biological macromolecule such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin, Refers to any foreign or unfavorable molecule, including at least one additive that may be present in the sample comprising the target molecule separated from the above. Additionally, such impurities may include any reagents used in the steps of the production and / or purification process. The leach and / or the extract are also impurities.

The term "purified," "isolated, " or" isolated, " as used interchangeably herein, means increasing the purity of a target molecule by separation from a composition or sample comprising a target molecule and one or more other ingredients, . Typically, the purity of the target molecule is increased by removing (completely or partially) one or more impurities from the composition.

The term "chromatography" refers to any sort of technique for separating an analyte of interest (e.g., a target molecule) from other molecules present in the mixture. Typically, the target molecule is separated from other molecules as a result of the rate at which the individual molecules of the mixture travel through the stationary medium under the influence of the mobile phase, or the difference in binding and elution processes. Examples for chromatographic separation methods are reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography and mixed mode chromatography.

A "buffer" is a solution that is resistant to changes in pH due to the action of the acid-base conjugate component. For example, various buffers that can be used depending on the desired pH of the buffer [Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffer, as well as combinations thereof.

A "disposable device" or "single-use device" means a product designed for a limited number of uses (either recycled after use or disposed of as solid waste) or, preferably, a product designed for disposable use For the purification of one batch of raw material produced from a bioreactor). The term often refers to affordability and short-term convenience rather than mid- to long-term durability. The term is also used in products that can last for weeks or months (eg, disposable filters) to distinguish them from similar products that last indefinitely (eg, washable filters). Preferably, the "disposable device" is used only once and the duration of use is defined as the duration of the method in which it is used, for example, the filtration method, the bioreactor method, and the like.

Disposable devices include, but are not limited to, plastic materials such as polyamides, polycarbonates, polymethylpentenes, polystyrenes, polyethylenes, polyesters, polyvinyls such as polyvinyl chloride, polysulfones such as polyethersulfone, polytetrafluoroethylene, Vinyl acetate or polypropylene. Disposable devices may be any device necessary for the production and purification of target molecules, such as bioreactors, full tanks, surge tanks, surge backs, any type of tubing, valves, columns, filters, cartridges or connectors.

The term "bioreactor " as used in the present invention refers to a manufactured or engineered device or system that supports a biologically active environment. In some instances, the bioreactor is a vessel in which a cell culture process is carried out that includes an organism or a biochemically active substance derived from such an organism. This process may be aerobic or anaerobic. A commonly used bioreactor is typically cylindrical in size from liters to cubic meters and is often made of stainless steel. In some embodiments described herein, the bioreactor may comprise a disposable ingredient made of a material other than steel and is disposable. In some embodiments, it is a disposable bag in which the biologically active environment is maintained. The total volume of the bioreactor may be any volume from 100 mL to 10,000 liters or less, depending on the particular method. Disposable or disposable bioreactors provide an alternative to reusable bioreactors and are preferably used to perform one biologic or biotechnological method before being discarded. By providing a new disposable bioreactor for each method, and preferably a disposable bioreactor that is sterilized during the manufacturing process, the complete cleaning and sterilization of the previously used bioreactor is performed while reducing the risk of (cross-) contamination Eliminates the need to record. Disposable bioreactors are often designed as containers with a flexible container, e.g., a bag, or at least a wall whose cross-section is flexible. Examples of such bioreactors are described in US 2011/0003374 A1, US 2011 / 0058447A1, DE 20 2007 005 868U1, US 2011 / 0058448A1, US2011 / 0207218A1, WO 2008 / 088379A2, US 2012/0003733 A1, WO2011 / 079180 A1, US 2007/0253288 A1 , US 2009/0275121 A1 and US 2010 / 0028990A1.

As used herein, the term "full tank" refers to any container, canister, reservoir, tank or bag, which is typically used between process steps and which is capable of collecting the entire volume of product / Volume. The full tank may be used to maintain or store or manipulate the solution state of the entire volume of product from the process step.

In some embodiments, methods and systems for the preparation and purification of target molecules can use one or more full or surge tanks during the process.

The term "surge tank " as used herein refers to any container or barrel or bag used between process steps or within process steps (e.g., when a single process step comprises more than one step) ; Where the output from one stage flows through the surge tank to the next stage. Thus, the surge tank differs from the full tank in that it is not intended to maintain or collect the entire volume of the product from the stage; But instead allows the product to flow continuously from one step to the next.

As used herein, the terms " purify, "" purification," and "purification step" refer to the removal of suspended particles and / or colloids, Refers to a method step for reducing turbidity. Purification can be accomplished by a variety of means including centrifugation or filtration. Centrifugation may be performed in batch or continuous mode, while filtration may be performed in steady flow (e. G. Deep filtration) or tangential flow mode. In processes used in industry today, centrifugation is followed by a deep layer filter, which is intended to remove insoluble impurities that may not typically be removed by centrifugation. Furthermore, as a method for improving the purification efficiency, for example, precipitation may be used. Precipitation of impurities can be carried out by various means such as flocculation, pH adjustment (acid precipitation), temperature transfer, phase change due to stimulus-reactive polymer or small molecule, or any combination of these methods. In some embodiments described herein, purification includes any combination of two or more of centrifugation, filtration, deep filtration, and precipitation. The term "deep filter" or "deep filtration" as used herein refers to a filter that can retain particulate matter through the filter medium, rather than merely on the filter surface. In some embodiments described herein, one or more deep filters are used in the purification method step.

The term " leach and / or extract " as used herein means an impurity that can be found in a liquid comprising the target molecule preparation or the target molecule during and / or after the preparation and / or purification method, And the apparatus used in the purification process, especially the disposable apparatus. An extract is a chemical compound that migrates to a liquid containing a target molecule or a target molecule from any material that is in direct contact with the target molecule when exposed to a suitable solvent under the conditions, time, temperature and mechanical strength of the particular condition. Substances capable of releasing the extract include elastomers, plastics or coating components.

The leachate may be removed from any target molecule-contacting material (including elastomer, plastic or coating components) as a result of direct contact with the liquid containing the target molecule formulation or the target molecule under conventional method conditions or accelerated storage conditions, A chemical compound that migrates into a preparation, typically a subset of the extract, found in a liquid containing the target molecule, and is also found in the final target molecule product if not removed.

Typically, the extract test is performed using a model solvent, while the leachate study uses the actual target molecule or process fluid.

Typically, the extract is obtained under exaggerated or aggressive conditions, but leachate testing uses conventional method conditions.

As a result, typically, the leachate is represented as a subset of the extract. In some cases, due to the interaction of the process fluid or method molecules with the method molecules, some leach compounds are not part of the extract.

Details of the extracts and leaching are described in BPSA Extractables and Leachables, BioProcess Int. 2007 , 5 (11) , 36].

Examples of leaches and / or extracts are acetaldehyde, toluene, 2-hexanone, acetone, 2-butanone, ethyl acetate, bisphenol A, benzyl alcohol, trimethylsilanol, formaldehyde, bis (2-ethylhexyl) 2-pentanone, 3,3-dimethyl-2-butanone, 3-ethoxy-propane, 3-hexane Butanol, tetrahydrofuran, and methyl formate. Examples of the organic solvent include methanol, ethanol, isopropanol, butanol, butanol, butanol, cyclohexanone, ethanol, formic acid, heptaethylene glycol, hexanel, methylisobutylketone,

The state-of-the-art biopharmaceutical manufacturing method typically uses a genetically engineered cell to express a target target in a bioreactor and performs a number of unit operations to purify it. In the case of some biopharmaceutical molecules such as monoclonal antibodies, the steps of ultrafiltration / diafiltration to obtain centrifugation, deep filtration, chromatographic purification, virus inactivation, chromatographic polishing, viral filtration and bulk drug substances Is used. Biopharmaceutical molecule purification methods have been established to remove potentially contaminated and deleterious agents such as bacteria, viruses, host cell proteins and host cell DNA, target molecule scaffolds or groups, purification method leaches and extracts (Shukla, AA, Gottschalk, Trends in Biotechnology, March 2013, Vol.31, No. 3, 147-154).

DETAILED DESCRIPTION OF THE INVENTION

The gist of the present invention is to find that activated carbon obtained by pyrolysis of activated carbon, especially organic polymeric material, can be used to remove leachates and / or extracts from the use of disposable devices. WO201404281 and US 2014/046038, it is generally known that activated carbon can be used to remove impurities in the biofarm preparation process. However, it has not yet been found that activated carbon is particularly suitable for removing leachates and / or extracts resulting from the use of disposable devices.

Activated carbon is a substance with a wide range of nonspecific adsorption properties and is used as an adsorbent or decolorizer in the industrial sector, such as in the manufacture of chemicals and food, sewage or wastewater treatment, water filtration, and the manufacture of low molecular weight drugs.

The term "activated carbon" or "activated carbon" as used interchangeably herein refers to a carbonaceous material that has been treated by a process that enhances its pore structure. Activated carbon is a porous solid with a very high surface area. They can be derived from a variety of sources including coal, wood, coconut shells, nutshells, peat and also organic polymers. Activated carbon can be prepared from these materials using physical activation with heating under controlled atmosphere or chemical activation with strong acid, base, or oxidizing agent. The activation process produces a porous structure with a high surface area that provides the activated carbon with high capability for impurity removal. The activation process can be modified to control the acidity of the surface.

It has been found that activated carbon obtained from organic polymers is particularly effective in removing leachates and / or extracts according to the present invention. The organic polymer is any synthetic, chemically defined organic polymer, such as, for example, polystyrene, polyamide, polycarbonate, polymethylpentene, polyethylene, polyester, polyvinyl or polypropylene.

Preferably, the activated carbon comprises or preferably consists of spherical activated carbon particles. This means that they have essentially similar extensions to all three spatial dimensions. Cubes, parallelepipeds or cylinders, except that the expansion to two different spatial dimensions is not more than three times, preferably less than two times.

Activated carbon obtained from organic polymers can be prepared by pyrolysis of spherical organic materials such as polystyrene. However, [Int. J. Electrochem. Sci., Vol. 4, 2009, pages 1063 to 1073], it is also possible to pyrolyze the glucose solution. The preparation of spherical activated carbon is further disclosed in US 20060148645 and US 2008171648.

An exemplary method of making such activated carbon polymer particles is to use polymer balls, particularly ion exchanger balls, polymeric structures (containing detachable functional groups, especially sulfonyl and / or carboxyl groups) as an educt. The porous polymer balls are pyrolyzed and the optionally pyrolyzed polymer balls are applied to the activation step. The separation of the functional groups preferably takes place at a residual content of 5% to 15% (based on the weight fraction of functional groups used). The temperature of the first heat treatment is suitably in the range of 200 DEG C to 350 DEG C for 10 minutes to 60 minutes. The atmosphere is, in principle, arbitrary. The following pyrolysis step essentially starts at a temperature corresponding to the final temperature of the first heat treatment and preferably ends at 600 ° C to 800 ° C. The heating rate is suitably in the range of 5 K / min to 0.5 K / min, from which the duration of the pyrolysis step can be calculated immediately. The activation step is not critical and occurs in a conventional manner.

Suitable spherical activated carbons are also available as SARATECH (TM) 100562, SARATECH (TM) 100772 and SARATECH (TM) 101373 (Blucher GmbH, Erkrath, Germany).

The activated carbon preferably has a surface area of 10 to 10 000 m 2 / g, more preferably 100 to 5000 m 2 / g, and most preferably 1000 to 2000 m 2 / g.

The average particle size of the activated carbon is preferably at least 2 [mu] m, more preferably from 2 to 550 [mu] m, very particularly from 5 to 40 [mu] m.

The properties of the particles are well known in the art and are preferably prepared by sieving. This is [I. C. Edmundson, Particle-size analysis, H. S. Bean, A. H. Beckett and J. E. Carles (eds) in: Advances in Pharmaceutical Sciences vol. 2, Academic Press, London 1967, 95-174.

The average particle size of the product fractions was determined using the EDANA recommended test method No. < RTI ID = 0.0 > WSP 220.2-05 "Particle Size Distribution ", wherein the mass fraction of the screen fraction is plotted in cumulative form and the average particle size is determined on the graph. Where the average particle size is the value of the mesh size resulting in a cumulative 50 wt%.

For example, the proportion of activated carbon having a specific particle size range of 5 to 40 탆 is preferably at least 90% by weight, more preferably at least 95% by weight, most preferably at least 98% by weight in the total activated carbon.

The type and amount of leachables and / or extracts that can be found in the preparation and / or purification methods of the target molecules depend on various aspects, such as the apparatus used in the method, as well as the conditions under which the method is carried out, such as temperature, . Typical leachates and / or extracts are selected from the group consisting of acetaldehyde, toluene, 2-hexanone, acetone, 2-butanone, ethyl acetate, bisphenol A, benzyl alcohol, trimethylsilanol, formaldehyde, bis (2-ethylhexyl) Propane, 3-hexanone, 2-butanone, 2-pentanone, 3,3-dimethyl-2-butanone, 3-ethoxy- , 3-methoxy-1-butanol, butanal, cyclohexanone, ethanol, formic acid, heptaethylene glycol, hexanal, methyl isobutyl ketone, pentanal, t-butanol, tetrahydrofuran, methyl formate. In particular, toluene, 2-hexanone, acetone, 2-butanone, ethyl acetate, bisphenol A, benzyl alcohol, trimethylsilanol, formaldehyde and bis (2-ethylhexyl) phthalate have been identified as important leachates and / or extracts . Toluene, 2-hexanone, acetone, 2-butanone and ethyl acetate have proven to be reference compounds suitable for monitoring and referencing process quality and amount of residual leachate and / or extract.

In order to carry out the method of the present invention, at any stage of the preparation and / or purification of the target molecule, the target molecule, typically present in the liquid, is contacted with activated carbon. This, of course, should be done after the method step in which the disposable device is used. Biopharmaceutical production typically involves several process steps such as cell culture, purification, chromatography, virus removal, filtration in a bioreactor. The preparation of the media and buffers used in these steps as well as in each of these steps may involve the use of a disposable device. Disposable devices may be used once or several times in manufacturing and / or purification methods. The use of activated carbon may be carried out once or several times in the production and / or purification process. This can be done as an additional method step, or it can be included in an existing method step, for example a filtration step.

In one embodiment, a target molecule, typically present in a liquid, is contacted with activated carbon after a method step in which the disposable device is used in combination with one or more of the following method features:

- Elevated temperature - this means that in this method step where the product solution and the disposable device are in contact, the temperature is at least partially above 25 ° C. This is the case, for example, when a disposable bioreactor is used in which the cells are typically cultured at 37 占 폚.

- Long exposure time - this means that the contact between the product solution and the disposable device in this method step is over one hour. For example, in the case of a disposable bioreactor in which cells are typically cultured for several days, or chromatographic separation takes longer than 1 hour, and a solution containing the target molecules is stored in a disposable tank for more than one hour. In the case of.

- Mechanical deformation - This means that the disposable device is mechanically deformed or treated mechanically (eg when using a hose pump or when the plastic bag is pressed together to release liquid contained therein). These modifications typically increase the release of leachate and / or extract from the plastic material.

In another embodiment, the target molecule, typically present in the liquid, is contacted with the activated carbon more than once in the process.

Contact with activated carbon can be performed by mixing a liquid containing the target molecules with activated carbon followed by separation of the liquid from the activated carbon, for example by sedimentation, centrifugation and / or preferably filtration. The contact time of the activated carbon with the liquid is typically 5 to 30 minutes.

In a preferred embodiment, the contacting is carried out by flowing a liquid comprising the target molecule through activated carbon. To this end, the activated carbon may be packed into a column, cartridge, filter or any other suitable device. Typically, the residence time of the liquid in the apparatus is 0.5 to 5 minutes. One of ordinary skill in the art can adjust the flow rate to achieve an appropriate residence time as well as the amount of liquid and activated carbon.

Liquids containing the target molecules are typically liquids resulting from the process steps after carrying out the process of the present invention. It may be water, buffer, organic liquid or any mixture thereof. Typically this is an aqueous buffer. The liquid typically contains not only the target molecule but also one or more impurities such as leachates and / or extracts.

Removal of leachates and / or extracts by activated charcoal can be carried out under a wide range of extraction conditions, such as conductivity and ionic strength. Typically, solutions containing target molecules can be applied directly to activated carbon without changing the pH, conductivity and ionic strength of the solution. The pH of the liquid containing the target molecule may be, for example, from pH 3 to 9 when it is contacted with activated carbon. Nevertheless, the presence of an organic solvent in a solution can affect the binding ability of the activated carbon.

Extraction of leachates and / or extracts with activated carbon is based on adsorption size exclusion, which means that leachates and / or extracts are bound to the pores of the activated carbon whereas the target molecules are too large to enter the pores, . Thus, the target molecule can be recovered in the non-adsorptive fraction and the leachate and / or extract is adsorbed on the activated carbon to reduce the amount of leachate and / or extract in the liquid comprising the target molecule.

The amount of activated charcoal used in the process of the present invention will depend on the amount and / or origin of the leachables and / or extracts that may be present. Typically at least 1 g of activated carbon is suitable for 0.005 g of leachate and / or extract. Typically 1 g of activated carbon may be used for 0.0001 to 0.005 g of leachate and / or extract, preferably 0.0005 to 0.0015 g of leachate and / or extract. In the case of toluene, acetone, and bisphenols, it has been found that the amount of activated carbon required to remove them from a solution containing the target molecule is less than that required to effectively remove the aldehyde. Preferably at least 1 g of activated carbon or more preferably about 1 g of activated carbon is used for about 0.001 g of leachate and / or extract such as toluene, acetone, bisphenol and about 0.0005 g of leachate and / , ≪ / RTI > for example acetal aldehyde.

It has been found that activated carbon obtained from organic polymers is particularly suitable for the removal of leachates and / or extracts. The most effective reduction of leachates and / or extracts can be achieved with this type of material.

It has further been found that activated carbon having a relatively small particle size of 4 to 50 [mu] m is particularly suitable for the process of the present invention.

The method of the present invention first provides an easy and effective method of removing leachates and / or extracts from target molecules. Activated carbon is a material known in the art that can be easily incorporated into purification processes without the risk of adding new contaminants to the target molecule.

The method of the present invention is suitable for removing at least 50%, preferably at least 75%, most preferably at least 90% of the leachate and / or extract from the target molecule. It has been found that the method of the present invention is particularly suitable for removing leachates and / or aromatic hydrocarbons such as extracts such as ketones, alcohols, toluene and benzyl alcohol from the target molecules. Appropriate device scaling is expected to remove 90% of the named leachate and / or extract.

The entire disclosure of all such applications, patents, and publications, cited above and cited below, in particular, Daewoin European Patent Application EP 14003737.5 (filed November 6, 2014) is incorporated herein by reference.

Example

Static combination of leachate and / or extract using activated carbon material

Adsorba® 150C hemoperfusion was used for the following experiments (see, for example, Activated Carbon 100772, Bluher GmbH, Erkrath, Germany ("BL772"); Activated Carbon 100562, Blucher GmbH, Erkrath, Germany Merck KGaA, Darmstadt, Germany ("Lichrolut"); Merck KGaA, Darmstadt, Germany ("Lichrolut"); Activated Carbon, Gambro Dialysatoren GmbH, Hechingen, Germany ("DVB particles")) were dried in a vacuum oven at 40 DEG C for 24 hours, and 1 gram of dry material was weighed into a glass flask. Then, 15 ml of water in which 0.1 g / L of the test molecule was dissolved was placed in a glass flask. The glass flask was then applied to the shaker for 15 minutes. After shaking, the glass flask was centrifuged at 4000 rpm for 15 minutes. The solution was then filtered and applied to headspace GC-MS. The value given is the mean value from three measurements (Figure 1-2).

The activated carbon materials ("BL 562" and "BL 772") were able to adsorb three of the four selected reference materials under the detection limit.

The activated carbon materials ("BL 562" and "BL 772") were capable of adsorbing two of the three selected reference materials under the detection limit and reducing the amount of acetal aldehyde approximately three times.

In addition, except for detection errors, an increased level of ethyl acetate (1 g / L) was used to flatten the adsorption capacity of activated carbon (Figure 1). The experimental setup was the same as described above, but the concentration of ethyl acetate was increased to 1 g / L and two activated carbon materials ("Bl 772" and "Gambro") were used (FIG.

The achieved results demonstrate that "BL 772" can successfully be used to adsorb ethyl acetate from the model solution.

Activated carbon and Millistak + CR40 filter for disposable bioprocessing assembly (Mobius Virus Clearance Assembly)

For the following examples, the disposable assembly was tested using pure water:

A 10 L solution bag of Mobius ® Disposable Assembly (inspected (at least 25-40 kGy; MS0010L30EP, EMD Millipore Corporation, Billerica, Mass., USA) was tubed with a Lynx ST connector (STC21THN01, EMD Millipore Corporation, Billerica, And a 10 L solution of Mobius Disposable Assembly (at least 25-40 kGy; MS0010L30EP, EMD Millipore Corporation, Billerica, Mass., USA) was connected to the Mobius Disposable Assembly (Pharma 50, 7486040801PU-6, EMD Millipore Corporation, A female lure (5621000821, EMD Millipore Corporation, Billerica, MA, USA) was connected;

The assembly was installed in a Mobius ® Virus Clearance unit (EMD Millipore Corporation, Billerica, Mass., USA) and 10 L of pure water was flowed at a rate of 1.5 L / hour using a unit peristaltic pump. The treated water was collected at room temperature in a 10 L solution bag of Mobius Disposable Assembly.

The 10L water-containing bag was then connected to an equilibrium Superformance® glass column (10 * 150 mm, Gotec Labortechnik, Bickenbach, Germany) packed with activated carbon (100772, Blucher GmbH, Erkrath) and centrifuged at 4 ml / min Lt; / RTI > The flow through the water was collected in a separate glass vessel and analyzed by FIA-MS (Bruker Esquire 3000+, Bruker Corporation, Billerica, MA, USA) at 200 μl / min using flow assisted ionization mass spectrometry (ESI + Respectively. The results are shown in FIG.

Filtration of the disposable assembly treated water through a glass column packed with activated carbon reduced the level of extract / leach (according to FIA-MS-ESI + method) to <ppm and exhibited a very rapid break through profile.

The bag containing 10 L treated water was connected to a balanced Millistak + media (EMD Millipore Corporation, Billerica, Mass., USA) in the uPOD (TM) format CR40 23 cm 2 containing activated carbon and pulverized at 4 ml / min Lt; / RTI &gt; Flows through the water were collected in individual glass vessels and analyzed by FIA-MS (Flow Injection Mass Spectrometry, 200 μl / min, Bruker Esquire) using electrospray ionization (ESI +, ESI-) and atmospheric pressure chemical ionization 3000+, Bruker Corporation, Billerica, MA, USA). The results are shown in Figures 5 - 8.

Filtration of the disposable assembly-treated water through the Millistak + ® CR40 filter reduced the level of extract / leach (FIA-MS-ESI +; depending on the ESI-method) to a strength of 10X.

Filtration of disposable assembly-treated water through the Millistak + ® CR40 filter reduced the level of extract / leach (corresponding to FIA-MS-APCI-, APCI + method) corresponding to MilliQ water quality.

Thus, the use of the Millistak + ® CR40 device for extract / leachate removal after using the disposable assembly almost completely removed the extract / leachate.

Applied activated carbon and Clarisolve filter for disposable bioprocessing assembly (Mobius ® Chromatography Assembly)

For the following examples, the disposable assembly was tested using pure water:

A 20 L solution bag of Mobius Disposable Assembly (at least 25-40 kGy; TF20020LGE1, EMD Millipore Corporation, Billerica, MA, USA) was tubed with a Lynx ST connector (STC21THN01, EMD Millipore Corporation, Billerica, MA, USA) Pharma 50, 7486040801 PU-6, EMD Millipore Corporation, Billerica, Mass., USA) and mounted on a Smart Flexware Assembly for Chromatography (At least 25-40 kGy; TF20020LGE1, EMD Millipore Corporation, Billerica, MA, USA) using a female lure (5621000821, EMD Millipore Corporation, Billerica, MA, USA) EMD Millipore Corporation, Billerica, MA, USA);

The assembly was installed in a Mobius Chromatography unit (EMD Millipore Corporation, Billerica, Mass., USA) and 20 L of pure water was flowed at a flow rate of 2 L / hour using a unit peristaltic pump. The treated water was collected at room temperature in a 20 L solution bag of Mobius Disposable Assembly. The bag containing 20 L water was then connected to an equilibrium Superformance® glass column packed with activated carbon (100772, Blucher GmbH, Erkrath) (10 * 150 mm, Gotec Labortechnik, Bickenbach, Germany) min. &lt; / RTI &gt; The flow through the water was collected in a separate glass vessel and analyzed by FIA-MS (Bruker Esquire 3000+, Bruker Corporation, Billerica, MA, USA) at 200 μl / min using flow assisted ionization mass spectrometry (ESI + Applied. The results are shown in FIG.

Filtration of the disposable assembly treated water through a glass column packed with activated carbon reduced the level of extract / leach (according to FIA-MS-ESI + method) to <ppm and did not exhibit a break through profile.

The 20 L water-containing bag was then connected to an equilibrium Millistak + media (EMD Millipore Corporation, Billerica, MA, USA) in uPOD ™ format CR40 23 cm 2 containing activated carbon and operated at 4 ml / min using a peristaltic pump. Flows through the water were collected in individual glass vessels and analyzed by FIA-MS (Flow Injection Mass Spectrometry, 200 μl / min, Bruker Esquire) using electrospray ionization (ESI +, ESI-) and atmospheric pressure chemical ionization 3000+, Bruker Corporation, Billerica, MA, USA). The results are shown in Figures 10 - 13.

Filtration of the disposable assembly-treated water through the Millistak + ® CR40 filter reduced the level of extract / leach (FIA-MS-ESI +; depending on the ESI-method) to a strength of 10X.

Filtration of disposable assembly-treated water through the Millistak + ® CR40 filter reduced the level of extract / leachant (FIA-MS-APCI-, APCI + method) corresponding to MilliQ water quality.

Thus, the use of the Millistak + ® CR40 device for extract / leachate removal after using the disposable assembly almost completely removed the extract / leachate.

Claims (13)

Wherein the target molecule is separated from the leachate and / or the extract by contacting the activated carbon with a liquid comprising the target molecule. The method according to claim 1, wherein the target molecule is a protein having a molecular weight of 10,000 or more. 3. The method according to claim 1 or 2, wherein the target molecule is a glycoprotein. 4. The method according to any one of claims 1 to 3, wherein the target molecule is an antibody. Process according to any one of claims 1 to 4, characterized in that the leachate and / or the extract is one or more of the following components: acetaldehyde, toluene, 2-hexanone, acetone, 2-butanone, ethyl Acetate, bisphenol A, benzyl alcohol, trimethylsilanol, formaldehyde, bis (2-ethylhexyl) phthalate, 1-methylethyl ester acetic acid, 2,4- Butane, butane, cyclohexanone, ethanol, formic acid, heptaethylene glycol, hexane, hexane, heptane, octane, Methyl isobutyl ketone, pentanal, t-butanol, tetrahydrofuran, methyl formate. 6. The method according to any one of claims 1 to 5, wherein the contact of the target molecule with the activated carbon is carried out at a pH of from 3 to 9. The method according to any one of claims 1 to 6, wherein the activated carbon is activated carbon obtained by pyrolysis of an organic polymeric material. 8. A process according to any one of claims 1 to 7, characterized in that the activated carbon has a median particle size of 5-40 [mu] m. 9. A process according to any one of claims 1 to 8, characterized in that the target molecule is separated from the leachate and / or the extract by filtration through at least one filter comprising activated carbon. 10. Process according to any one of claims 1 to 9, characterized in that it is used in the production and / or purification process comprising one or more of the following steps:
- Cell culture in a bioreactor
- Purification
- chromatographic purification
- Filtration. 11. The method according to any one of claims 1 to 10, wherein the target molecule is selected such that the disposable device is used in combination with a mechanical deformation of the disposable device at a temperature in excess of &lt; RTI ID = 0.0 &gt; 25 C & Characterized in that it is contacted with activated carbon after a method step of more than one hour. Use of activated carbon for removing leachates and / or extracts from a liquid. 13. Use according to claim 12, characterized in that the liquid is contacted with activated carbon in a filtration device.

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